This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Background &Aims: Identify and characterize the complete Identification and characterization of the CD4 epitopes are important not only for the development of effective vaccine for AIDS virus infection but also help understand other diseases pathogenesis. Despite the enormous progress on basic immunology and epitope mapping strategies, the general property of a functional T cell epitope is not yet fully understood. Consequently, accurate screening strategy to identify the complete epitope repertoire is not available either. Moreover, understanding the property of functional T cell epitopes is crucial to understand cellular immunity, mechanism of viral evasion and will provide a rationale approach for improving AIDS vaccine design. In the studies described in this application, we propose to elucidate and identify the properties and characteristics of the functional CD4 T cell epitopes. More importantly, to develop a simple and accurate system to screen the complete antigen-specific CD4 epitope repertoire restricted to a specific MHC class II molecules in the rhesus monkey model. If the CD4 epitope-screening method is successfully developed, not only the scientific community that use the monkey model will be beneficiated but also could be directly apply in the human system. Our hypothesis is that the major characteristic of a functional T cell epitope is to have high binding affinity to the MHC class II proteins enough to bind as a long peptide fragment during the antigen processing steps. The hypothesis is based on data generated using our novel peptide binding affinity assay using the TCR tetramer for the CTL epitopes. The analysis of five well characterized SIV specific CTL epitopes restricted to the Mamu-A*01 molecule with consistent characteristics and common properties indicated that the TCR tetramer technology could be easily apply for the identification of the CD4 T cell epitopes. Finally, since the MHC class II peptide holding groove is wider compared to the MHC class I molecule, we also hypothesize that the number of available CD4 epitopes should be higher than the CTL epitopes. Specific Aims: 1. Construct a TCR tetramer specific for the p46/Mamu-DR*W201 complex. 2. CD4 epitope repertoire for the whole SIV protein restricted to the rhesus monkey MHC class II molecule Mamu-DR*W201 using overlapped pool peptides and the newly constructed TCR tetramer. 3. Identify the complete repertoire of functional CD4+ T cell population specific for the whole SIV virus from vaccinated monkeys. Thus, conforming the utility of the TCR tetramer for the screening of functional CD4 eptopes. Results: Unfortunately, due to the delay in recruiting personnel with the necessary skills to perform the proposed study, the entire project was considerably delayed. First, , a Medical Research Specialist was originally assigned to this project, however, she moved back to the Division of Microbiology soon after the grant was awarded. Finally, in April of 2008 Dr Yohei Saito with the necessary background in molecular biology has just joined as a postdoctoral fellow. Therefore, in April of 2008 we have requested a non-cost extension of this project and it was approved until 12/31/09. Aim 1. This study has finally actively started with the recruitment of Dr Saito. In order to select the appropriate TCR alpha beta sequences for the construction of the TCR tetramer, reassembling of alpha and beta chains from an oligoclonal antigen-specific T cell population into a functional antigen-specific TCR molecule will be necessary. Since April of 2008, Dr Saito has optimized the PCR amplification protocol for the TCR alpha-beta sequences. With his current protocol, the full length of the TCR alpha and beta sequence could be efficiently amplified from 104 sorted lymphocytes. Moreover, he has also succeeded in the amplification of the TCR alpha-beta sequences from sorted cells down to 102 cells following a pre-amplification step. This step has become important since the stocked RNA was isolated from only 103 sorted p46/Mamu-DR*W201 tetramer-binding T cells from two SHIV infected rhesus macaque monkeys. We are now in the process of cloning the full length TCR alpha-beta sequences specific for the p46/Mamu-DR*W201 complex. All possible TCR alpha-beta combinations will then be co-transfected into Schneider (S2) cells by calcium phosphate precipitation. In order to identify the correct functional TCR alpha/beta pairing, a large amount of the p46/Mamu-DR*W201 tetramer will be needed. Unfortunately, the originally described protein production system using the S2 cells failed to produce the necessary large quantity of stable p46/Mamu-DR*W201 monomers. With Dr Saito's expertise, we are now in the process of developing a new protein expression system adapted from the system used to produce large quantity of recombinant IgG proteins. The vectors used for the expression of the Mamu-DR*W201 monomers is described. The Mamu-DR-alpha and Mamu-DR*W201 chains has been already successfully cloned into the pCIRN Neo-R and pEF IRES GFP vectors, respectively. Both expression vectors will then be co-transfected to 293T cells lines and selected in the presence of neomycin. In order to isolate cells containing both plasmids, the culture cells will then be sorted for the presence of the GFP protein. Finally, the His-tagged Mamu-DR*W201 proteins present in the supernatant of the large scale culture of the isolated 293T cells will be purified by using the commercially available Ni-column. The positive pair of alpha and beta chains will then be selected to generate the soluble p46 TCR tetramer. Aims 2 and 3. These studies will start upon completion of the SA1. A new reagent constructed from the SA1 is a crucial component for the completion of these studies. Significance The development of an accurate and efficient approach to identify and screen the complete antigen-specific CD4 epitope repertoire restricted to a specific MHC class II molecules in the rhesus monkey model will allow us to better understand antigen-specific CD4 responses to help develop efficient vaccine against AIDS. Plans During the next year, with the addition of the postdoctoral research fellow with considerably background in molecular biology, we expect to start SA2 and SA3 of the proposed application.